ES2403155B1 - PROCEDURE OF PRODUCTION OF HAPLOID PLANTS, DOUBLE HAPLOIDS AND / OR DIHAPLOIDS, BY GYNOGENESIS - Google Patents

Abstract

The invention relates to a process for the production of H haploid, double HD haploid and / or DH dihaploid plants, the HD and DH being homozygous or essentially homozygous, this being a type of procedure that concerns the technique of pollen-induced gynogenesis. irradiated This method comprises an irradiation stage of the male parental reproductive material with a dose between 160 and 190 gamma rays and / or a stage of selection of haploid H and / or DH plants by using molecular marker (s) ). The invention also relates to a method of producing haploid, double haploid and / or homozygous dihaploid plants comprising a step of determining the appropriate irradiation dose (s) to increase the yields of said plants as a function of multiple factors given as the plant species, the genotypes of the male and female parental, climatic and physiological conditions, the time of harvest of the fruits, the level of growth of the embryos collected with a view to their cultivation, the level of development of embryos put in culture. On the other hand, the invention relates to the molecular marker (s) used in the selection stage as well as to haploid embryos, dihaploid embryos obtained by the process of the invention, the offspring and the seeds of the plants obtained by the process of the invention.

Description

PROCEDURE FOR PRODUCTION OF HAPLOID, DOUBLE HAPLOID AND / OR DIHAPLOID PLANTS, BY GYNOGENESIS

The scope of the invention is that of plant selection or improvement, that is, the production of plants both in the form of embryos and in any other stage, especially from the seedling to the adult plant.

More precisely, plant selection or improvement according to the present invention means obtaining a homozygous or essentially homozygous haploid or diploid plant with a stable offspring in terms of its phenotypic and / or genotypic characters. In other words, for a plant of this type, a genome fixation is achieved with a reduced number of generations (for example, one or two).

More precisely still, the present invention relates to a new method of producing plants (eg embryos, seedlings, adult plants), Haploides (H), Double Haploides (HD) and / or DiHaploides (DH), homozygous or essentially homozygous, this being a type of procedure that concerns the gynogenesis technique advantageously associated with pollen irradiation. The affected plants are, for example, zucchini.

Varietal creation systems, that is, the creation of new plants adapted to the specific needs of farmers and producers, have accelerated since the early twentieth century. One of the most drastic changes was the development of hybrids, also commercially called F1 hybrids, initially in corn, and the use of the phenomenon of heterosis, which corresponds to the increase in the capacities or vigor of a hybrid in numerous characters (vigor , performance, resistance to diseases and bedriddenness, prematurity) on the average of the two parents or on the best of the two parents. Another change refers to the use of in vitro culture techniques of plants based on their totipotence.

The creation of F1 hybrid plants, which in particular allow the dominant characters of their parents to gather in the F1 hybrid plant, quickly spread from corn to other species such as tomatoes, peppers,

eggplants Indeed, in addition to the hybrid vigor present in some species, obtaining F1 hybrids also allows an improvement in the homeostasis capabilities of the plant (plant and plant stability).

expression of their characters in different environments), and the possibility of accumulating genes of interest. However, the creation of F1 hybrid plants

It implies the creation of relatively homozygous parental lines. Once crossed, these parental lines allow obtaining the F1 hybrid.

While the genetic purity of the parental lines is maintained, the hybrids

F1 can be obtained repeatedly.

This search for homozygosis of the parental lines does not develop

only to obtain F1 hybrid plants. Similarly,

During the development of new plants marketed in the form of population varieties (lettuce, beans, miracles, etc.), the creation of

Relatively homozygous cultivars (or varieties) has become a necessity. The requirements of homogeneity of the product for farmers and for marketing (criteria of homogeneity and

stability of the new cultivars inscribed in the catalog), as well as the

mechanization and the increasing precision of cultivation techniques and

production implies the need for more and more homogeneous plants in the

Expression of your characters.

The coach traditionally reaches the level of homozygosis sought

self-fertilizing the most promising plants of several generations, selecting those that present the desired characters, progressively homogenizing the genome of the plants from one generation to another.

The new zucchini plants developed by the sorters are

Either autocult autocultivars, or F1 hybrids. In both cases, the search for homogeneity of the cultivar and / or the parental lines of the F1 hybrid is one of the objectives of the selection program.

Remember that to start the sexual cycle of plants, it is necessary

a process of reducing the number of chromosomes (meiosis) to give

origin to gamnetic cells with a haploid number of chromosomes (n). In flowering plants, sexed reproduction implies double fertilization. Pollen grain produces two gamnetic nuclei (n)

male or reproductive nuclei. A male nucleus fuses with an egg (n) to form a zygote (2n) that produces the embryo by restoring the

number of somatic chromosomes (2n). Another male nucleus (n) joins

with the polar nuclei of the haploid embryo sac to form a cell

triploid (3n). In some cases, zygote formation does not occur, but

the cell divisions of the ovum nevertheless begin, reaching a

Haploid embryo capable of giving rise to a seedling whose haploid genome is entirely of maternal origin (Sarkar and Coe, Genetics, 1966, vol. 54,453-464). Haploid plants exist in a small number in the

nature and are sterile.

The discovery, in the early 1920s, of viable haploid plants and the possibility of duplicating their genome has aroused

Numerous investigations Indeed, these haploid plants are interesting not only in the field of genetics, but also for the improvement

of the plants since, after chromosomal duplication (spontaneous or not), the

Genetic information is identical in the two chromosomes of each pair.

In this way, the genetic information is fixed and the double haploids allow to accelerate the selection processes.

In 1964, Guha et Maheshwari (Guha and Maheshwari, Nature, 1964, 204, 497) discovered that some plants can be regenerated from haploid cells during the cultivation of pollen grains.

Since then, numerous investigations have been launched about

of the production of haploid plants of various species, using

in vitro culture techniques of gametophytes. The two main techniques of

Production of embryos and haploid plants (haploidization) by in vitro culture of gametophytes are androgenesis and gynogenesis.

In androgenesis, immature male gametophytes are placed in

culture in a synthetic medium in order to obtain haploid embryos that then develop as whole plants. The results vary with the genotype, but also with the protocols used.

In gynogenesis are mature female gametophytes (ovaries or ovules)

those that are put in cultivation.

The two techniques can be used with the same species, knowing that one of them is more advantageous for certain species, while the other technique is better suited to other species.

Some authors also cite the production of embryos and plants

haploids through the use of chemical or physical agents. The principle of

This technique is to cause the development of the unfertilized egg in the plant (in situ). Although some attempts through technical shocks, X-rays or

chemical substances have only led to mediocre results, the use of irradiated pollen has given, in certain species such as melon,

results not directly applicable to other species.

The majority of plants induced by these techniques are haploid plants,

but other plants with varying levels of ploidies can be obtained. If aneuploids or tetraploids may present no more than interest

lower in plant improvement programs, spontaneous diploids (dihaploides OH) are very sought after since the spontaneous duplication of their genome during the first phases of in vitro culture makes them fertile.

These homozygous plants are directly usable by the sorters, which represents a huge advantage at different levels

(time, space, cost ...).

Haploid plants (H), which are obtained through these different

techniques and that have not spontaneously doubled their genome, they must suffer

then a supplementary stage to make them diploid (2n) or Double Haploides (HO). This can be done using different chemicals such as colchicine (alkaloid that allows duplication of a genome).

The double haploid HO, as well as the dihaploid OH (result of a

spontaneous diploidization) are homozygous individuals, which can be

Use directly as homogeneous cultivars (population varieties) or as parental lines of hybrid varieties. In fact, these HO and OH plants have, in duplicate form, the genetic information of a single set (n) of chromosomes, that of the female gamete from which they originally descended.

In this way, these in vitro plant creation techniques allow not only to gain time, but also to achieve a better genetic homogeneity: the genome is stabilized (homozygous) in a single generation instead of reaching genomic homozygosis after multiple generations of self-fertilization. . These also allow to improve the selection program

since they reveal the recessive characteristics of the plant created in this way. The use of double HD haploids and

dihaploides OH is therefore a very interesting tool. Its use has been extended to certain species: androgenesis is used for

plants of the genus Brassica (Keller et al., in K. Giles, S. Sen (eds.), Plant Cell Culture in Crop Improvment, 1984, 169-183. Plenum Pub. Corp., New York), gynogenesis for cucumber (European patent EP 0374755) Yla sugar beet (Hosemans and Bossoutrot, Z. Pflanzenzuecht, 1983, 91,74-77), pollen irradiated in melon (Sauton et Dumas de Vaulx, Agronomie 7,1987, 7 (2) , 141-148).

Consequently, the plants that have been used for the development of these in vitro haploidization techniques are commercially available today in various species and registered in the official catalog.

However, despite these commercial varieties, the yields of

the different haploidization procedures still depend too much on multiple unknown or not fully controlled factors (genotype,

cultivation of the mother plants, conditions of haploidization, etc.) and sometimes they are still too weak, in numerous species, to be routinely integrated into the current selection procedures.

With regard to Cucurbita pepo zucchini, only some fragmentary results are known today and none indicates how to produce enough H haploid embryos (given below by double HD haploids), or DH dihaploid embryos to maintain an improvement program vegetable.

The first mention of the in vitro culture of unfertilized Cucurbita ovules

But Dumas de Vaux et Chambonnet made it in 1986 (Dumas and Chambonnet, Genetic Manipulation in Plant Breeding, 1986, 285). The ovules

They are removed from the ovaries one or two days before flowering or that morning. In the first two cases, the flowers do not pollinate, in the third, they are pollinated, but not fertilized. The eggs are placed in Petri dishes in a medium that contains macro or micronutrients, as well as vitamins,

2,4 dichlorophenoxyacetic acid (2.4 D) Yquinetina. The authors obtain their best performance, that is 4.3 embryos per 100 cultured ovules, when

The ovaries are removed one day before flowering. However, they find

difficulties in obtaining plants that develop normally. The origin

of the plants is analyzed during a later stage of self-pollination of

fertile diploid plants and during a phenotypic examination of the

offspring.

More recently, in the case of Cucurbita pepo zucchini, Shalaby (Shalaby, Scientia Horticu / turae, 2007, 115, 1-6) analyzed the influence of the genotype, the position of the female flowers on the stem, the temperature

and of the sucrose concentration during gynogenesis. Regenerated haploid plants are treated with colchicine in order to obtain double

haploid The authors conclude that the genotype used greatly influences

measured in the possibilities of using this technique routinely. From

this way the Yellow Bik genotype does not produce any embryo by

gynogenesis, while responding to androgenesis.

The conclusion of this article regarding the influence of the genotype indicates

clearly that the techniques of androgenesis and gynogenesis are not satisfactory today to be used routinely and routinely in

a program to improve the zucchini Cucurbita pepo.

It has been proposed to irradiate pollen in a gynogenesis procedure

induced to obtain haploid H plants, and / or DH dihaploid plants, giving haploid H plants then double HD haploid plants. The irradiation of the pollen grain causes it to lose its fertilizing power (the DNA of the gametes is damaged by radiation), but retains its germinative power, thus being able to induce the division of the oosphere and its development in embryo.

This technique of gynogenesis with pollen irradiation is especially used

in melon (Cucumis me / o) (Sauton, Cinquantenaire de la Culture in vitro, 24-25 October 1989) where most genotypes are capable of producing embryos and / or haploid or dihaploid plants.

In the technique of gynogenesis applied to zucchini Cucurbita pepo and referred to by Kurtar et al. (Kurtar et al., Euphytica, 2002, 127, 335-344), pollen irradiation is also used. Kurtar et al. have studied the influence of the

irradiation dose and genotypes in obtaining embryos

haploid These authors obtain embryos and haploid plants without having been able to optimize the irradiation dose and having found that the

Embryo production is strongly influenced by the level of irradiation, by the development of embryos put in culture and by the

genotypes Thus, contrary to what happens with melon, the technical conditions of production of haploid embryos of Cucurbita pepo zucchini cannot, so far, be generalized.

This multifactor variability constitutes a major first drawback of known gynogenesis procedures, with or without irradiation of the

pollen.

A second drawback of these procedures has to do with the analysis of the state of ploidia and the state of homozygosis of the plants obtained (embryos / seedlings / adult plants). Indeed, the plants may be haploid or dihaploid, but these may have different levels of ploidia, in particular they may be haploid-diploid, tetraploid or aneuploid chimeras (Dumas de Vaux and Chambonnet in 1986).

In the opposite way to the principle of gynogenesis, irradiation of the pollen can lead to fertilization of the ovule by the DNA of the pollen grain, mainly by DNA fragments. This may be due to the fact that the radiation has not sufficiently degraded the DNA to prevent

partial fertilization, which results in the formation of a chimeric embryo (without wanting to be limited by theory, these inventors think of

recombinations between the ovule DNA and pollen grain DNA fragments). When the radiation degrades the DNA enough, the pollen grain

he should lose his fertilizing power and keep only his power

germinative It has been found, however, that in some cases, along the length of the pollen tube, some fragments of male DNA

they can be in contact with the ovule and, during a

pseudo-fertilization, recombine with the female DNA, thus giving plants

haploid chimeric (when at recombination between the ovule's DNA and the

DNA fragments from irradiated pollen grain are followed by DNA loss

leftover during the following cell divisions) or with different ploidies,

being able in particular to be heterozygous in certain genes when they have

one or several supernumerary chromosomes. Haploid chimeric plants are interesting because they have only one copy of the information

genetics (even if a very small part of the genome comes from the male gamete) and because a duplication of its genome, just as in

haploid, produces homozygous or essentially homozygous plants.

Some diploid heterozygous plants can also be produced

resulting from a "classical" fertilization, in the case, for example, of insufficient irradiation.

In addition, in the cucurbitaceae, it has been found that some plants

obtained by means of these different in vitro culture procedures can be diploid but heterozygous, because they do not undergo gamma reduction

during the formation of female gametes (Veilleux, P / ant Breeding Reviews, 1985, 3, 253-288). In this way they have all of the

genetic information of the plant of origin and therefore do not present the

characteristics of interest sought and characteristic of haploid, HD and / or DH plants.

Known methods to determine ploidy levels allow

differentiate haploid plants n from diploid plants 2n. But nevertheless,

current methods to more accurately determine the level of

ploidy (chimerism, chromosomes or fragments of chromosomes that are missing or supernumerary) are still very expensive in materials and time.

Thus, a simple, fast and economical method would be necessary to differentiate between haploid and chimeric plants resulting from

A pseudo-fertilization. In addition, there is currently no satisfactory option to differentiate 2n heterozygous diploid plants obtained by fertilization, 2n heterozygous diploid plants resulting from fertilization, 2n heterozygous diploid plants resulting from non-gammatic reduction and homozygous 2n dihaploid DH diploid plants

resulting from spontaneous duplication.

The identification of H haploids (chimeras or not) and of the different diploids can be done by cytological analysis, especially by chromosome counting in preparations of root cells of analyzed plants

in the optical microscope. Cytological analysis is difficult to apply in a

Varietal creation program where speed and accuracy are necessary.

This does not solve, however, the problem of haploid identification.

regarding non-haploid chimeras resulting from a pseudo-fertilization.

The selection of diploid 2n plants is done so far by phenotypic analysis of the offspring of the regenerated plant; the

Self-pollination of a homozygous plant should give a stable homogeneous offspring throughout successive generations of self-fertilization. If this is not and a segregation of characters is observed, it is because the regenerated plant was not homozygous, but

heterozygous Thus, until now it has only been possible to identify the DH dihaploids by morphological selection and / or the use of recessive markers (for example, liguleless, glossy ...)

present in the female parental genome. The use of these recessive markers has the disadvantage of needing, prior to

the induction of gynogenesis, its introduction in the genome of the line

intended to be used as a female parent, if it does not already have them. Some coloring markers can be used in the selection of

haploid or dihaploid embryos and plants (Chase and Nanda, Maize Gen Coop, 1965, News letter, 39, 59-60; Coe and Sarkar, J of Heredity, 1964, vol. 55, 231-233). However, the expression of these markers is influenced by the female parental genotype or even by the general process of embryo maturation and this can lead to an evaluation.

Incorrect origin of the individuals obtained.

The phenotypic analysis of the offspring is long, since it is necessary to wait for flowering, obtain the grains and observe the offspring of each

grain in order to verify its homogeneity.

The causes of the absence of satisfactory results in the different

Known haploidization methods are, in part, at the limits

current in vitro culture techniques, that is, the great specificity

genotypic response in in vitro culture, the low percentages of differentiation to give rise to embryos, the significant losses of these embryos when you want to achieve a complete organogenesis of these.

There is, therefore, no satisfactory technique to date.

produce reproducibly enough plants (eg embryos / seedlings / adult plants) H haploids, double HD haploids and / or dihaploides

OH, in order to maintain a selection program, in particular of

Zucchini Cucurbita and particularly Cucurbita pepo is part

indeed of these species in which the effort deployed is very

important to overcome different problems.

In this context, one of the objectives of the present invention is to provide an effective process that allows to improve the production of

H haploid plants, double HD haploids and / or DH dihaploids, by

gynogenesis induced by an irradiated male gametophyte (for example,

irradiated pollen).

Another objective of the present invention is to provide a new improved process of plant selection or improvement, that is, of

obtaining a homozygous, haploid or diploid plant or essentially

homozygous with a stable offspring in terms of its phenotypic and / or genotypic characters, or, in other words, a plant for which genome stabilization is achieved with a reduced number of

generations (for example, one or two).

Another object of the present invention is to provide a method of

production of haploid, double haploid and / or dihaploid plants, by

Ginogenesis induced with irradiation of male gametophytes (pollen), solving the drawbacks of the prior art and satisfying at least

one of the following specifications:

-

Optimization of the irradiation dose;

-

Be able to free, at least in part, from the influence of the level of development of embryos in culture and / or genotypes and / or conditions

climatic and / or the time of harvest of the fruits, the level of

growth of collected embryos with a view to their culture, among other variables (multifactor variability); -Generalization of the procedure to a maximum of species and genera, in particular to most Cucurbita pepo zucchini; - Allow an effective and safe analysis of the state of ploidy and the origin of the plants produced; - Allow a good identification of the plants obtained.

Another object of the present invention is to provide a method of

production of haploid plants (which then give double haploid plants) and / or dihaploides, by irradiated pollen-induced gynogenesis, allowing said procedure to reduce the time, as well as the

cultivation space necessary for creation and varietal selection, thanks to

a stage of selection of haploid plants, and / or dihaploides, from among the

set of plants produced with different ploidies.

Another object of the present invention is to provide a method that

allow, beyond a process for producing haploid and / or dihaploid plants by gynogenesis with irradiation of the reproductive material of the

male parental, determine the appropriate irradiation dose to increase the production yields of these plants based on

multiple factors given and preferably selected from among the plant species, the male and female parental genotypes, the

climatic and physiological conditions of these plants, the time of harvest of the fruits, the growth conditions of the immature embryos collected with a view to their cultivation.

Another object of the invention is to provide at least one of the following

elements:

-Molecular marker (s); -Embryos; -Regenerated plants from embryos; -Outline of plants; -Seeds of plants;

that are usable in or obtained by a production process of

haploid, double haploid and / or diplohaploid plants, by gynogenesis

Induced by irradiated pollen, this being a type of procedure as indicated in the objectives mentioned above.

Another object of the invention is to provide an effective method of analysis of the origin of plants, especially of plants produced by a method of producing haploid plants (which then give double haploid plants) and / or dihaploids, by gynogenesis induced by

irradiated pollen, this being a type of procedure like the one stated in the objectives mentioned above, and that allows to determine if the plants contain genetic material of the male parent.

Another object of the invention is to provide an effective method of

identification of plants, especially of plants produced by a

Haploid plant production procedure (given below

double haploid plants) and / or dihaploides, by gynogenesis induced by

irradiated pollen, this being a type of procedure like the one stated in the objectives mentioned above and that allows to determine if

Plants are homozygous or heterozygous.

At least one of the foregoing objectives, among others, is achieved with the present invention which concerns, first of all, a new process

of production of haploid H, double haploid HO and / or dihaploid OH plants, the HO and OH being homozygous or essentially homozygous, being

This is a type of procedure that corresponds to the technique of gynogenesis induced by irradiated pollen, characterized in that it comprises:

-

an irradiation stage of the male parental reproductive material with a dose between 160 and 190 gamma rays, preferably between 165 and 185 gamma rays (Gy);

-

or a step of selecting haploid H, or OH, or H and OH plants by using a molecular marker;

-

or an irradiation stage of the male parental reproductive plant material with a dose comprised between 160 and 190 gamma rays, preferably between 165 and 185 gamma rays (Gy) and a selection stage of haploid plants H, or OH, or H and OH through the

use of a molecular marker.

It should be noted that in this application, normally the article

undefined "a" should be considered as a generic plural (meaning "at

minus one "or even" one or more "), except when the context indicates what

opposite (1 or "only one"). So, for example, when previously

It says that an irradiation stage is added, it is the addition of one or several irradiation stages. The same happens with the stage of plant selection that can be done by several stages of selection, with the use of one or several molecular markers.

The process according to the present invention opens a new way of

Obtaining plants (lato sensu) haploid H, double haploid HO and dihaploides OH. This new way brings improvements in terms of:

-expansion of the spectrum of vegetables affected by this technique of

ginogenesis with irradiation of male gametophytes (pollen); -reduction of the multifactor variability characteristic of this technique so far; -analysis of the state of ploidia of the plants produced; -identification of the origin of the plants obtained; -reduction of time and cultivation space necessary for creation and

varietal selection.

On the other hand, this procedure has proved totally adequate for

Zucchini plants.

Given the factor variability of pollen irradiation-induced gynogenesis, all advances in terms of optimization are welcome. Thus, according to another of its aspects, the invention points to a

production process of haploid H plants, double haploid HO and / or dihaploides OH, the HO and OH being homozygous or essentially

homozygous, by irradiation-induced gynogenesis of the material

male parental breeder, characterized in that it comprises a previous stage of determining the (or the) adequate irradiation dose (s) to increase the yields of obtaining said plants based on multiple factors given and selected preferentially from among the species vegelal, parental genolipos

male and female parental, climatic and physiological conditions, the

moment of the harvest of the fruits, the level of growth of the embryos

collected with a view to its cultivation.

The invention also points to a plant production process

H haploids, double HD haploids and / or DH dihaploids, the HD and DH being

homozygous or essentially homozygous, by irradiated pollen-induced gynogenesis, characterized in that it comprises the use of a molecular marker to identify plants free of male parental genetic material whose pollen has been irradiated or possessing an insufficient amount thereof to cause, in the offspring of these plants, a disjunction of a phenotypic and / or genotypic character as well as to determine

his homozygous state.

According to another of its aspects, the invention relates to a molecular marker that can be used in the process according to the invention, this marker being selected, for example, from microsatellite markers.

(SSR), defined by the pairs of attached nucleotide sequence primers SEO ID No. 1 to 16, preferably SEO ID No. 3 to 14.

According to another of its aspects, the invention relates to haploid H embryos,

HD and / or DH of plants, as intermediate products of the process according to

the invention.

According to another of its aspects, the invention relates to the offspring of the plants obtained by the process according to the invention.

According to another of its aspects, the invention relates to the seeds of

plants obtained by the process according to the invention.

In order to better understand the invention, it is first of all worth remembering or giving some definitions, which should be considered in any case as

non-exclusive examples.

-

Genotype means the set of genetic material that an individual carries and that constitutes their inheritance. A genotypic character corresponds or not to a phenotypic character, which can be recessive or dominant.

-

The term phenotype designates the set of morphological characters or

apparent functional functions of an individual, which in turn correspond to the expressed part of the genotype and to some phenomena determined by the environment.

-

A plant means a plant in any stage of development in which it is found, in particular: embryo, or any other stage seedling or of the

adult plant

-

By male or parental male plant is meant a plant that is used as a pollen donor in a cross.

-

By female or parental plant is a plant that is used as a recipient of pollen or as an egg donor at a cross.

-

Male reproductive material means the male flower or any part that includes the male haploid gamnetic cells, that is, the

etamina, male gametophytes or pollen grains.

To simplify, in the present application, pollen means the male reproductive material used.

-

Female reproductive material means the female flower or any part that includes the female haploid gamnetic cells, the ovaries, the ovules, the female gametophytes (embryo sac) or the cells

female reproductive (oosleras).

-

Autógama means a plant whose mode of reproduction is autogamy and whose grains are obtained from the fertilization of two gametes

extracted from the same individual.

-

Pollination means the contribution of a grain of pollen from the stamina (male organ) to the female organs. Pollination gives

place to a fertilization, even a pseudo-fertilization when the male material used has been irradiated.

-

Fertilization means the fusion between a male gamete and a female gamete giving rise to an embryo.

-

Pseudo-fertilization means the induced formation of an embryo without prior fusion of a male and female gamete. In the field of

present invention, the irradiation-induced gynogenesis technique of

Pollen allows the formation of an embryo by pseudo-fertilization. Pseudo-fertilization can also be a partial fertilization, when

some DNA fragments of the male gamete fuse with the gamete

female.

-

Self-fertilization means the pollination of an individual by its own pollen, and the resulting individuals are called self-fertilized.

-

Hybrid plant means a plant resulting from the cross between two genetically different parents. For a first generation plant, a cross between two homozygous parents

genetically different, for example, two different varieties, we can talk about F1 hybrid.

-

Heterosis or hybrid vigor means the phenomenon by which an F1 hybrid is significantly superior to the best of its parents compared to one or

several characters, especially as regards vigor.

-

Genome means the number of chromosomes contained in the nucleus

of the cell, or the amount of DNA.

-

By "allele" it is understood according to the present invention the various versions of a given DNA sequence located in a given chromosome locus (location on a chromosome).

-

Homozygous means a cell or an individual that has two identical alleles of the same gene in a given locus of the same pair of chromosomes, and this for the characteristic provided by that gene.

-

Essentially homozygous means plants according to the invention, when the identity of two alleles is verified for example by at least one

80%, preferably 85%, in particular 90% and more particularly for 95% of the alleles examined or when after self-fertilization, there is no segregation of the characters in the offspring. By extension, in the present application, essentially homozygous plants are considered homozygous plants.

-

Diploid means a qualifier applicable to cells or plants or parts of plants that comprise this type of cells, which have,

at its core, a batch of similar chromosomes two by two, called

counterparts Diploid cells are usually the result of the fertilization of two haploid gametes.

-

Haploid means a qualifier applicable to cells or plants or parts of plants that comprise this type of cells, whose chromosomes

Its core contents are each only a single copy (n).

-

Chimeras means a qualifier applicable to cells or to plants or parts of plants that comprise this type of cells, which contain a

Additional AON fragment from irradiated pollen or from which one of the chromosomes has undergone recombination with the AON of irradiated pollen. These cells can be haploid H, dihaploides OH (when there is spontaneous duplication of the genome) or with different ploidies.

When a plant is a chimera with different ploidies (especially comprising chromosomes or fragments of supernumerary chromosomes), it is heterozygous for certain alleles.

When a plant is obtained from a recombination with a fragment of

AON of irradiated male pollen, this is interesting in two cases:

1) If you have no more than a single copy of each of the chromosomes, it is considered as haploid and therefore may undergo duplication.

Posterior chromosome to become a homozygous double haploid (2n) plant.

2) If you have suffered a spontaneous chromosomal duplication, this is homozygous (2n) dihaploid.

.

-

Double haploid (HD) means a qualifier applicable to cells or to

plants or parts of plants that comprise this type of cells, whose genome has been artificially multiplied, most of the time by chemical treatment and mainly by colchicine.

-

By dihaploides (DH) is meant a qualifier applicable to cells or to plants or parts of plants that comprise this type of cells, these cells being haploid at the beginning and that have seen their genome doubled

spontaneously. This duplication of the genome allows to obtain a cell,

plant or part of plant with 2n completely homozygous.

-

Hybrid female parental means a hybrid plant that is used

as a pollen receptor plant in a cross.

-

Zucchini means a plant of the genus Cucurbita. Zucchini is actually a group of cultivars of the Cucurbita pepo species and the subspecies Cucurbita pepo ssp. pepo.

-

By dose is understood the dose of irradiation absorbed by the target, that is,

in particular the reproductive plant material of the male parent.

-

By molecular marker is meant a specific fragment of a

DNA sequence that can be identified inside the genome of an individual and that can be used in particular to locate a gene of interest, verify if an individual has inherited a particular characteristic of a parent or differentiate two individuals. It may or may not be a coding sequence. The marker can be dominant or co-dominant. The detection of the molecular marker, or its non-detection, allows the selection of

individuals presenting the gene of interest or the particular characteristic, or by

On the contrary, do not select individuals who do not present the gene of interest or the particular characteristic. In the present invention, the markers

Molecules make it possible to quickly test plants or seedlings during their development and choose those that have the characteristics

wanted. The person skilled in the art knows molecular markers of

different types: AFLP (amplification fragment length polymorphisms), SCAR (amplification product characterization) SSR (microsatellites, single sequence repeats), RFLP (restriction fragment length polymorphisms), etc.

-

By microsatellite marker or microsatellite sequence or also SSR is meant a DNA sequence formed by a continuous repetition of

motifs composed of 1 to 4 nucleotides. These microsatellite sequences are present in the genome as a whole, most commonly at the level of

introns of genes, but also at the level of exons. The microsatellite polymorphism can be used as a genetic marker in order to identify an individual or a population, for example, plants.

In the creation and selection of plant varieties the transmission of

stable and homogeneous way of the selected characters is a

indispensable prerequisite. It is also essential to know in a way

Reliable genotype and phenotype of plants produced. To keep a

hybrid, preferably an F1 hybrid, parental lines are necessary

stable. In order to obtain fixed plants, the

plants repeatedly, the plants not presenting the characteristics sought by being eliminated in each generation. New techniques have allowed the

plant development from gametes only (n). Thus, androgenesis and gynogenesis have developed. The use of gynogenesis

With irradiated pollen it is commonly used to generate plants. But nevertheless,

As previously stated, this technique presents some

limitations and mainly cases of pseudo-fertilization by pollen, even when it has been fragmented by radiation, preferably

X-ray or gamma Indeed, it may happen that the pollen has not degraded enough and that a DNA fragment of the irradiated pollen

«Pseudofecunde» the female gametophyte, while in this technique

It should only induce the development of a haploid embryo without modifying its genome. This embryo, and consequently the resulting plant of this

embryo, in the best case should not contain genome of the male parental that has been used for pollination of reproductive material

of the female parental. But it turns out that the offspring produced by irradiated pollen-induced gynogenesis can be of different types, in particular:

-

haploid planlas (with n chromosomes),

-

dihaploid plants (with 2n identical n + n chromosomes), -chimeras containing n female parental chromosomes and some fragmented pieces of recombined or not recombined male parental DNA

with the AON of the female parent, either in haploid form, or with different ploidies, or being dihaploids that have undergone spontaneous duplication of their genome,

- diploid plants resulting from fertilization, - Diploid plants that have the same genome as the mother plant (2n).

In the processes of varietal creation, it is necessary not to conserve more than

haploid H plants (including haploid chimeras), which can then duplicate the genome of double HO haploids, and dihaploid OH plants to be sure of their homozygosis.

After long research, researchers have perfected this selection by proposing a carefully chosen range of irradiation doses of male gametophytes, which allows to guide gynogenesis

towards a population of desired individuals (H, OH) exempt or almost exempt from

unwanted individuals.

The fact of associating or replacing the choice of this dose range with a selection process through the use of molecular markers and / or

Fiujo's cytometry goes in the same direction.

According to a preferred mode of application of the invention, the process in question of radiation induced gynogenesis of the pollen comprises the following successive steps:

a) Apply male parental reproductive material, preferably a flower; b) Irradiate said male parental reproductive plant material with a dose between 160 and 190 gamma rays, preferably between 165 and 185 gamma rays (Gy) and more particularly between 170 and 180 Gy; c) Pollinating the reproductive plant material of the female parent with said irradiated male parental plant material; d) Collect fruits whose grains contain embryos; e) Extract the grains of said fruits;

f) Remove the embryos from said grains;

g) Put the embryos in culture in a suitable medium until obtaining a plant, preferably a seedling.

The irradiation stage of the male reproductive material can be repeated

several times, preferably two or three times, so that the material

Genetic is quite degraded. The irradiation dose of the especially preferred male reproductive material is around 175 Gy.

In a particularly preferred mode of application of the invention, the

The procedure also involves, after step g) already described above, a step h) which consists in selecting the haploid H and / or OH plants by flow cytometry, or by using molecular marker (s), or by flow cytometry and use of molecular marker (s).

This stage h) of selection of haploid H and / or OH plants involves:

-I-Ia use of specific molecular marker (s) of given allele (s) contained in the genetic material of the irradiated male parent to select from among the plants obtained after stage g ) plants that do not contain irradiated male parental genetic material or that have an insufficient amount of this to cause in the offspring of said plants, a disjunction of one or more phenotypic and / or genotypic characters;

-11-the use of specific molecular marker (s) of given allele (s) contained in the genetic material of the hybrid female parent, allowing this (these) marker (s) to determine the homozygous / heterozygous state of the plants to be selected, that is, the plants obtained after step g), this (these) marker (s) being used to select the homozygous and essentially homozygous plants, in particular the OH plants.

It is evident that during the h) stage of selection, the order of use of the specific molecular markers given contained in the genetic material of the male parent and of the contents in the genetic material

of the female parental matters little. I and 11 are not necessarily performed in

a precise order and can be concomitant (1 and 11 at the same time) or successive (1 and then 11, or 11 and then 1). In addition, the selection stage h) can

Repeat several times. This can be interesting when 5 several specific molecular markers are used to make successive selections increasingly accurate with fewer and fewer individuals to test.

Finally, when stage h) consists of selecting the haploid H and / or OH plants by flow cytometry and use of molecular marker (s), flow cytometry may precede the use of marker (s)

10 molecular (s), the use of molecular marker (s) may precede flow cytometry, and the use of molecular marker (s) may be

concomitant to flow cytometry.

Preferably, the parental plants are selected from among the genus Cucurbita, family of the cucurbitaceae, preferably among the species Cucurbita pepo, and, even more preferably, among the subspecies Cucurbita

pepo ssp. pepo.

According to a remarkable feature of the invention, it is applied as a plant

20 female of hybrid plants and F1 hybrid plants. Indeed, during the production of gametes from a hybrid plant or from a plant

F1 hybrid, the chromosomal pairings of homologous chromosomes

during the first division they allow genetic recombinations and give

consequently a great genetic variability of the gametes produced. The

25 haploid H plants (which give the double haploid HO) and the dihaploid OH plants resulting from these gametes and which are obtained by the

The method according to the present invention is genetically different from each other, thus offering a greater choice to the selector.

To briefly review the life cycle of flowering plants, such as

Zucchini, a pollinated or self-pollinated flower will produce fruits that in turn contain grains, which contain embryos, which are going to develop in seedlings until they become plants.

The male and female plant material is selected based on the

characteristics sought. The male reproductive plant material

harvested and applied mainly comprises flowers of a male plant

selected, but it can also be ethamines or gametophytes or directly from male gametes or pollen grains.

The flowers for example are advantageously collected from the mother plant

male at the time of the anthesis. One or two are generally used

Male flowers for fertilization of each female flower. In the plants

preferred according to the invention, that is, those of the genus Cucurbita, the male plants chosen according to the invention may be, for example, those whose

Genotypes are in the group comprising: JIB, JEOIOA, ESKENOARENI.

These male plants are of special interest because they are good

pollinators, that is, they emit significant amounts of pollen.

The timing of the harvest of the male parental reproductive plant material, preferably flowers, may be important. This moment is

choose depending on the development of the plant from which the flowers come from, from

the station ... So, it is preferable to collect them during the anthesis and

Fertilize the female flowers before they open.

The irradiation stage b) of the parental reproductive plant material

male, that is, of male gametophytes, such as pollen, is

important for the success of the production process of H plants (which give double HO haploids) and / or OH plants.

The rays used are preferably gamma rays, but X-rays can also be used.

Irradiation is performed using known and suitable means such as

gamma rays, for example, for a sufficient time for the reproductive plant material of the male parent to receive the prescribed dose in jars

closed containing preferably ten to twelve male flowers per

jar. For example, for JIB, the irradiation time is preferably between 20 minutes and an hour and a half.

The chosen irradiation conditions (without distinction between parameters)

they allow to obtain very satisfactory results, that is, after pollination with the male reproductive plant material, a sufficient production of fruits, which contain a sufficient number of embryos that are

they become plants of which most possess the characteristics sought. In fact, most of the embryos and, consequently, of the plants produced by the process according to the invention are H and / or DH, the haploids giving double haploids.

Before performing step c) of the process according to the invention, you can

it is advantageous to germinate some irradiated pollen grains, in a

suitable medium, such as in a medium called MP15 (15% sucrose solution, 100 mg / L H, BO, and 700 mg / L CaCI "2H, O), in order to

verify that irradiated pollen still has germination capacity. After a

given germination time, for example 15 to 60 min, pollen grains

They are then observed through the magnifying glass in order to determine the

percentages of sprouted grains and open grains.

The female parental reproductive material used will have a genotype

selected according to the plant to obtain.

In preferred plants according to the invention, that is, those of the genus

Cucurbita, the female plants chosen according to the invention may be by

example those whose genotypes are in the group of genotypes that include VN001, VG06, VN002, Odessa, VG 14, Tosca et VE 547 also

can be used

Step c) of the process according to the invention consists essentially in the pollination of the female parent's reproductive material with the material

male parental player.

In this gynogenesis technique, whole plants can be regenerated from

of the female gametophytes (ovaries or ovules) contained in the reproductive plant material of the female parent.

'5

Pollination is advantageously in contacting the male gametes and the female gametes, but since the male gametes are irradiated there can be no real fertilization of the female gametes but only an elongation of the pollen tube and, by induction

of the oosphere, the development of an embryo.

Pollination of the female parental reproductive plant material can

Be done in different ways. Preferred techniques will be pollination by the "flower to floD" technique in which the entire surface of the stigmas

(end of the pistils) is coated with the parental reproductive material

masculine, but also by brush pollination. In the technique «flower a

fiaD> a legit or female parent's reproductive material is

«Fertilized» by a flower or by the male parental reproductive material.

In the so-called "pinceb> technique, a brush is used to perform the

transfer of pollen grains from parental reproductive material

male to one or several flowers or reproductive material of the female parent.

Advantageously, in the days following the pollination, the correct formation of the fruit (curdled) is observed and the fiories that do not form fruit are eliminated. After pollination, the time after which the fruits are harvested depends on the family, the genus and the species in question.

Preferably, fruits whose maturity is suitable for embryo extraction are harvested.

The morphology of the fruit and its external appearance allows to evaluate the opportune moment to gather the fruit.

For example, around 4 or 5 weeks (variable time according to gender and

species) after pollination, the fruits are harvested and preferably

then washed with water and possibly with a detergent (eg Marseille soap), rinsed, dried and eventually cleaned of

New with alcohol.

The fruits now open to extract the grains, preferably in

sterile conditions, for example, with laminar flow.

The beans are then opened to remove the embryos. stm !!! .. 91

The extracted embryos are placed in culture in an adapted medium, of the type described in the examples that we will see later, being

S2P medium is preferred (see table 8).

Embryos, for example, are grown in the dark, one or two nights, at

20-25 oC, then put in full light for 10 hours to approximately 20 hours, p. ex. About 16 hours at the same temperature.

Embryos can be collected at different stages of development

embryonic and some stadiums can influence the performance of the

procedure according to the invention. Indeed, embryos can be collected in the globular embryonic stage, heart, torpedo or cotyledon.

Embryos can be replanted regularly, preferably every 15

days approximately, in the same medium or in a different culture medium

adapted to its stage of development, particularly from the moment of

appearance of young leaves and small roots.

When the seedlings are well developed, they are replanted advantageously in another suitable culture medium, whose composition is p. ex. the one given in the examples that we will see later (see tables 8 and 9).

In a preferred variant of the process according to the invention, a step of selecting the haploid H and / or OH plants produced according to the process is carried out:

h1) by flow cytometry and / or, h2) by using a molecular marker.

This selection h1 and / or h2 is advantageously made when the plant resulting from gynogenesis presents some leaves, being extracted and

analyzing a sample of the plant, preferably of leaf, according to this

selection stage h) of the procedure.

These selections h1) and / or h2) that can be applied, simultaneously or not,

in any order, even discontinuously, they allow especially

determine the level of ploidy of the plants produced. Through these

techniques it is possible to distinguish between plants that have 2n chromosomes and plants with n chromosomes. Thus, plants that have n chromosomes (haploid H) are preserved to then undergo a stage of duplication of their genome, preferably with colchicine,

thus producing double haploids (HD). Haploid H plants are sterile, while double Haploid HD plants and DH dihaploids are not and

They are of special interest to the selectors.

In the case of selection h1) by flow cytometry, flow cytometry allows a reliable distinction between 2n plants and n plants, but this technique does not allow differentiating DH (2n identical, homozygous) dihaploid plants and plants 2n heterozygous diploids resulting from a defect of gamnetic reduction or fertilization by pollen grain. During this selection, both 2n plants and plants will be preserved

n.

The 2n plants will be analyzed later with markers

molecular (stage h2 already mentioned) in order to make a more precise selection between homozygous or essentially homozygous 2n dihaploid plants and heterozygous 2n diploid plants.

N haploid plants (chimeras or not) will be preserved with a view to

2.

also analyze by molecular markers, with a view after

obtain double homozygous or essentially homozygous haploids after

genome duplication

Flow cytometry selection allows plants to be classified into two

groups: haploides n and dihaploides 2n.

As alternative or complementary methods to flow cytometry, the chromosome count performed at root tips or counting can be cited

of the number of chloroplasts in stomatal guard cells that are

Perform in the optical microscope.

The selection h2) by means of the use of a molecular marker offers the possibility of distinguishing between the different types of 2n plants and

exposed above. This allows a very important time, space and cost savings. In addition, this method of selection also offers a much more precise analysis since it also allows to determine if the plants

Haploid H or dihaploides OH contain or not genome of the male parent, and this reliably and quickly. In this way, the selection method by using a molecular marker allows to determine the

homozygous status of the plant in question, but also its level of

ploidy

The h2) selection by using a molecular marker is

preferably a step h2) of selection of the H and / or OH plants that includes:

-1-the use of a specific molecular marker of given allele (s) contained in the irradiated male parental genetic material to select from among the plants obtained after step g) plants that do not contain genetic material of the irradiated male parent or having an insufficient amount to cause in the offspring of said selected plants a disjunction of a phenotypic and / or genotypic character;

-11-the use of a specific molecular marker of given allele (s) contained in the genetic material of the hybrid female parent, allowing this (these) marker (s) to determine the homozygous / heterozygous state, possibly the ploidia level, from plants to

select, that is, the plants obtained after step g), using this

(these) marker (s) to select homozygous or essentially homozygous plants, in particular OH plants, making it clear that at least one marker used for each analysis can be a

single marker, during a single analysis, especially in the field of a codominant marker that allows to immediately distinguish between several allelic forms of the same genetic marker. Preferably, a specific molecular marker of at least two alleles is used.

This specific molecular marker of allele (s) of the irradiated male parental genetic material and / or one, preferably two, given alleles contained in the hybrid female parental genetic material is

preferably an SSR microsatellite marker.

These different markers therefore serve, on the one hand, to identify plants that have a part of male genetic material in their genome and, on the other hand, to differentiate homozygous plants from

heterozygous plants. The more molecular markers are used, the more

the level of homozygosis is defined. It may happen that in a given locus the plant is homozygous with the molecular markers used,

but that is not homozygous (or essentially homozygous) in the set of

your genes It is therefore preferable to use several molecular markers

(preferably at least 3, particularly 5 and more particularly 6) for

reliably select OH plants. These markers are selected based on the genomes of the parents used in gynogenesis.

These molecular markers make it possible to distinguish between homozygous haploid H and / or dihaploid OH plants that have resulted from female gametes and heterozygous diploid (2n) plants resulting from a non

gametic reduction, resulting from fertilization, or recombined resulting from partial fertilization with irradiated pollen.

The markers are advantageously selected so that they identify different alleles between the female plants that give the female gametes and the male plants that give the pollen that radiates. In this way, a plant resulting from partial fertilization possesses the alleles from the two parental plants (male and female) while a haploid H or dihaploid DH plant has no more than the alleles from the female mother plant. The plants resulting from the gynogenesis induced by irradiated pollen and that have male parental alleles in their genome that confirm a "fertilization" are therefore discarded. The

Haploid chimeras are a particular case since they have alleles from the female plant and the male plant, but each allele

It is only present with only one copy.

In short, if the female plant contains the A and B alleles and the male plant contains the e alleles, all regenerated plants that have an e allele are

considered as resulting from a fertilization and are therefore

discarded Only plants that have only the A allele or the plants that have only the B allele are preserved. Plants that have the A allele can be AA plants when they are dihaploid and A

when they are haploid, the plants that have the B allele can be

BB plants when they are dihaploides and B when they are haploid. In the particular case of haploid chimeras, their identification may be possible.

by using several markers.

This (these) molecular marker (s) may also serve to classify and discard diploid plants resulting from non-reduced gametes and identical to the mother plant, since these are heterozygous for given and determined alleles of the female material used (mother plant). In this case, the molecular marker (s) is selected so that it identifies the alleles found in the heterozygous state in the female plants and, consequently, also in the

plants resulting from non-reduced gametes but found either

in the homozygous state in the case of dihaploid plants, or with a single copy in the case of haploid plants.

In summary, if the female plant contains the A and B alleles, all the regenerated plants that carry the AB allele combination are considered to be the result of a non-reduced gamete. Here also only the plants that have only the A allele or the plants that have only the B allele are chosen.

According to a preferred form of the invention, microsatellite markers or SSRs are used to determine, in the plants obtained and tested, both the presence of the male parental genome and the state

homozygous / heterozygous. They can also be used to determine the level of ploidia of the plants obtained and tested.

According to a particular and remarkable form of the invention, the molecular labeling (s) used for the application of the method according to the invention, is (are) a microsatellite marker (s) (s) ). Each one of

these microsatellite molecular markers are defined by a sequence

given nucleotide of a given size, amplified by PCR thanks to a pair of specific primers (a sense primer and an antisense primer). For the selection stage h), the molecular marker (s)

that can be used in the present invention are the eight markers

following: CMAGN73, CMBR153, CMBR22, CMMP73, CUCNITRA, FAD2, PATL1, T J3. Each of these markers is defined by a pair of nucleotide primers of given sequences (see SEO ID No. 1 to 16 above). It is clear that for this stage h) markers can be used

molecular (microsatellites or not) other than the eight markers mentioned above. Preferably, the markers defined by the

primer pairs SEO ID No. 3 and SEO ID No. 4; SEO ID No. 5 AND SEO ID No. 6; SEO ID No. 7 AND SEO ID No. 8; SEO ID N ° 9 AND SEO ID N ° 10; SEO ID No. 11 AND SEO ID No. 12 and / or SEO ID No. 13 AND SEO ID No. 14.

Sequences SEO ID N ° 1 to 16 are particularly interesting for the

application of the method of the invention. In addition, the invention also points to a molecular marker that can be used in the process.

according to the invention, of the microsatellite type (SSR), and selected in a way

advantageous within the group of microsatellite markers defined by the

pair of attached nucleotide sequence primers SEO ID No. 1 to 16.

The invention preferably points to a molecular marker susceptible to

used in the process according to the invention, of the microsatellite type, and

advantageously selected within the group of microsatellites defined by the primer pairs of nucleotide sequences attached SEO ID N °

3 to 14.

To increase the reliability of the selection, it is preferable to use a combination of two molecular markers according to the invention, that is, preferably microsatellite markers. Even more preferably according to the invention, a combination of three microsatellite molecular markers, particularly four microsatellite molecular markers, is used, and more particularly a combination of five microsatellite markers is selected. Obviously more than five molecular markers can be used if necessary. For these combinations of microsatellite markers used, the sequences

of the pairs of primers that define the marker (s) are selected from the sequences SEQ ID No. 1 to 16, preferably SEQ ID No. 3 to

14.

The SEO ID No. 1 to 16 microsatellite markers that can be used are in particular specific to the Cucurbitaceae family,

preferably of the genus Cucurbita and even more preferably of the

Cucurbita Pepo species.

The microsatellite molecular marker (s) (SSR) of zucchini

according to the invention, they are selected according to plant genotypes

for irradiated pollen induced gynogenesis.

This (these) microsatellite molecular marker (s) is defined by its

nucleotide sequence to be detected by amplification by reaction in

string (PCR) using specific primers whose sequences SEQ ID 1 to

16 are detailed in the examples that we will see below. These primers

they can be fragments of cloned polynucleotides or chemically synthesized oligonucleotides. Sequence markers SEQ ID No. 3 to 14 will preferably be used.

The plants selected and / or obtained after stage h are haploid H and dihaploid DH plants. H plants give DH dihaploid plants when genome duplication is spontaneous or double HD haploids

when genome duplication is done by chemical or physical treatment,

preferably using colchicine.

The process according to the invention therefore entails

preferably a stage i) of duplication of the genome of haploid H plants. preferably the stage of duplication of the

chromosome genome using colchicine.

HD plants and 2n are grown on land in a greenhouse while haploid H plants are preferably "mattified" in vitro and then replanted in a suitable medium, then rooted and grown on land in a greenhouse.

This genome duplication technique using colchicine comprises of

advantageously the following successive stages:

1-Isolate the main apex of the plant as well as axillary buds if there are and; 2-Prepare a colchicine solution, sterilize it below; 3-Place the cuttings in small sterile Petri dishes; 4-Cast a sufficient amount of colchicine to cover them, then close the Petri dishes; 5-Let it soak for several hours; 6-Remove the cuttings and rinse them in boats with sterile water; 7-After having dried them slightly on sterile paper, replant all cuttings in the middle of origin; 8-Replant the developing buds in the environment of origin; 9-When the cuttings have taken root, cultivate them in vivo on a support with A nutritious solution

As explained above, the present invention extends beyond the production of H, HD and DH plants by gynogenesis

induced by irradiated pollen. In effect, it proposes a new method of

plant production optimization. This method allows solving the problem of multifactor variability and generalizing gynogenesis with

irradiation of pollen to numerous plant species.

This method is based on a previous stage of determining the (or the)

adequate irradiation dose (s) to increase yields of

obtaining the plants, taking into account multiple factors such as

plant species, the genotypes of the male and female parents, climatic and physiological conditions, the time of harvest of the fruits, the level of growth of the embryos collected with a view to their

culture.

This previous stage consists essentially in applying, for each dose of

irradiation under test, a step h ') of selection of plants H and / or OH, h') being the same as h) as defined above, to then establish the yields in corresponding plants and at the end

Determine the dose (s) that offer the best yields.

In addition to the yields in plants, in this previous stage also

They can extract other results such as: the percentage of fruits obtained, the

percentage of grains obtained, the percentage of embryos and plants H, HD and OH, for a given irradiation dose.

Thus, the present invention is about a method of

production of haploid H, double haploid HO and / or dihaploid OH plants, the HO and OH being homozygous or essentially homozygous, by

ginogenesis with irradiation of the male parental reproductive material,

characterized in that it comprises a previous stage of determining the (or) adequate irradiation dose (s) to increase the yields of said plants based on multiple factors given and preferably selected from among the plant species, the parental genotypes

male and female parental, climatic and physiological conditions, the time of harvest of the fruits, the level of growth of the embryos collected with a view to their cultivation, the level of development of the embryos

cultivated positions; consisting of this previous stage:

i) in testing, for a given factor, different irradiation doses

on the reproductive plant material of the male parent,

ii) in applying, for each irradiation dose tested, a step h ')

of selection of the H and / or OH plants, including said stage:

-1-the use of a specific molecular marker of allele (s) of the irradiated male parental genetic material to select from plants obtained after irradiated pollen-induced gynogenesis, plants that do not contain irradiated male parental plant material or that they have an insufficient quantity to cause in the offspring of said plants a disjunction of a phenotypic and / or genotypic character;

-11-the use of a specific molecular marker of an allele,

preferably two specific given alleles of the genetic material of the

female parental hybrid applied in gynogenesis, allowing this marker to determine the homozygous / heterozygous state, possibly the

ploidia level, of the plants to be selected, that is, the plants obtained after gynogenesis, this marker being used to select homozygous or essentially homozygous plants, in particular OH plants.

Ii) in counting, for each irradiation dose tested, the plants

haploids H, or OH, or H and OH obtained;

iv) to calculate, for each irradiation dose tested and from

counts resulting from (iii), yield in haploid H plants, or

OH, or H AND OH obtained;

v) in comparing the counts resulting from (iii) and / or the yields resulting from (iv) to deduce the appropriate irradiation dose (s) therefrom.

As mentioned above, stage h 'involves the use of markers and the order of use of the molecular marker (s)

specific (s) of allele (s) of the male genetic material and the specific (s) of the allele (s) of the female material matters little. I and 11 no

they are necessarily performed in a precise order and can be concomitant

(1 and 11 together) or successive (1 and then 11, or 11 and then 1).

The invention also concerns the H and OH plants produced by the

procedure as defined above, knowing that the

term "plant" encompasses here all stages of development: embryos, seedling, adult plant. In particular, it concerns the haploid H and dihaploid OH embryos obtained, as intermediates, by the process according to the invention as defined above and the plants

regenerated from said embryos.

The invention also concerns HO plants and seedlings obtained by the process as defined above with step i) of

mattress

On the other hand, the invention also relates to offspring as well as to

the seeds and grains from the plants obtained by the

procedure as defined above. Indeed, this

application also refers to the offspring of the plants obtained by the procedure as defined above, obtaining said offspring by crossing said plants (HO or OH) between them or by crossing between a HO or DH plant obtained by the process so and

as defined above and a plant resulting from a line or resulting from repeated self-fertilization.

Finally, the invention also concerns the seeds of plants obtained by

the procedure as defined above or from the

embryos as defined above, as well as from

plants resulting from the offspring as defined

previously.

The preferred conditions of application of the procedures described above also apply to the other elements of the invention seen above, especially to the process of optimizing plant production by determining the appropriate irradiation dose, at

the embryos and the offspring of the plants obtained by the

procedures as well as its application.

The following examples illustrate the present application.

EXAMPLES

1-The manufacturing process of haploid plants H and dihaploides OH by irradiated pollen-induced gynogenesis

according to the invention.

The tests are carried out in greenhouses whose temperature at night is

of 15 oC and for the day of 25 oC.

The plants are carried in mulch bags (2 plants per bag in the case of the female and 3 plants per bag in the case of the male), on trellis and dripped watering.

The greenhouse is shaded and ventilated and pest control is done by integrated biological protection.

The plant biological material used consists of the following:

Female plants: 2 genotypes. VN001: 66 plants. VG06: 66 plants.

Male plants: 1 genotype. JIB: 90 plants.

Plants are labeled with a number and the name of their genotype.

Stage a)

On the eve of irradiation (or ionization) all male and female plants

they are checked and only the flowers that will be used for pollination are preserved. The number of male flowers in the appropriate stage (the end of the corolla begins to turn yellow) is counted to know approximately the number of pollinations that will be made for each dose

irradiation The same goes for female flowers in the stadium

adequate (the end of the corolla begins to turn yellow).

Some controls are prepared:

-

Traveler control (V): flowers travel in the same conditions but do not radiate;

3.

-

Non-traveler control not cut (NV): the collars are collected immediately before pollination.

Stage b)

The male collars are extracted very early in the morning (from 5:30 a.m. to 6:00 a.m.).

-

Flower conditions during transport and irradiation.

10-12 flowers are introduced into each jar, which will close. The closed jars are placed in a refrigerator with a refrigerant block. The treatment or type of control is noted on each jar. The irradiation of the reproductive material of the

Male parental is done in the morning for an hour. This will be repeated

on five different dates and two doses of irradiation will be tested:

dose H 175 gamma rays (Gy) dose O 200 gamma rays (Gy)

-

Estimation of germination power of irradiated pollen.

Pollen is germinated in a 45 ~ L drop of medium with 15% sucrose, 100 mg / L of H, BO, and 700 mg / L of CaCI "2H, O.

The drops are deposited on an EPOXY sheet and left 45 min in a Petri dish whose atmosphere is saturated with water, at 26 OC.

The percentage of sprouted grains and open grains is determined with the

magnifying glass.

Stage e)

Two hours after irradiation the reproductive material of the male parent is used for pollination.

A female fior or female parental reproductive material is fertilized with

a male flower or male parental reproductive material.

The days after pollination, the fruit set is verified and if

This is not produced, the fruit is eliminated.

Stage d)

The fruits are harvested 4 to 5 weeks after pollination and are

They label according to their genotype, the treatment of male flowers or the type of control.

The fruits are harvested preferably in the morning.

Stage e) Each fruit is washed with water and with Marseille soap, rinsed and dried. 70 'alcohol is poured under the laminar flow on the fruit that is cleaned

continuation throughout its surface with a sterile paper.

From this stage all operations will be carried out in conditions

of sterility under laminar flow.

An incision is made around all the fruit, then the

Pulp using a knife.

The beans are collected using a spoon, separated from the pulp with a clamp and a scalpel and then counted.

The beans are stirred overnight.

Stage f)

Under the laminar flow the wide jar is emptied into a tall sterile jar by a sterile funnel, then rinsed with sterile water. The beans are

they open one by one under the binocular loupe using a clamp and a scalpel.

The instruments are regularly sterilized to avoid any risk of contamination.

The embryo is deposited in an S1C culture medium, whose composition is

details in tables 8 and 9 that we will see later, in Petri dishes of

60x15, 4 embryos per Petri dish.

Embryos obtained after irradiated pollen fertilization do not have the

Same form as normal embryos.

Petri dishes are placed in the dark for 1 to 2 nights on camera at

20/25 oC, then they are brought to light in the same chamber at 20/25 oC for 16

hours.

Stage g)

The embryos are grown in the culture medium by the PPM (plant

preservative mixture) marketed by Kalys®.

The embryos are replanted in the S3P medium (P = PPM) whose composition is shown in Tables 8 and 9.

Petri dishes are checked regularly. The embryos are replanted if

there is contamination by a bacterium or fungus.

Embryos are replanted every 15 days in Petri dish in a medium

Again as long as they are not developed enough.

From the moment of the appearance of young leaves and small roots the embryos are transferred to a medium in S3P tubes (see composition in the

Tables 8 and 9).

Well-developed seedlings are replanted next in jar in S2P

or S4P whose compositions are listed in Tables 8 and 9.

Stage h)

From the moment that foliar development is enough, you take a

sample of each plant and analyzed by flow cytometry as well as by molecular markers SSR.

Stage h1)

A sample of each plant is taken and the level of ploidy is analyzed by flow cytometry according to the protocols developed by Laat et al. in Theo. Appl. Genet, 1984.67: 463-467 and in Plant Breeding, 1987, 99: 303

307. Plants with abnormal, undetermined ploidies are discarded and

Plants with n chromosomes differ from plants with 2n chromosomes.

Stage h2)

A sample is taken from each plant, the DNA is extracted according to the CTAB protocol modified by Tomas et al. (1989). The SSR marker (s) is selected from the SSRs defined by the primer pairs of SEO ID N ° 1 to SEO ID N ° 16 sequences that we will see later. The FAD2 marker is defined by the primer pairs SEO ID Na 11 and SEO ID No. 12. The PCR reaction is performed in a reaction volume of 5, .tI

consisting of half of 5 ~ I of diluted DNA, 0.85 ~ I of water, 1 ~ ll of 10x buffer, 1 ~ ll of MgCI2 (25 mM), 1 ~ I of dNTP (2.5 mM), 0 , 5 ~ I of each primer (2 ~ M), 0.075 of queue M13 (8 ~ M) and 0.075 of Taq Invitrogen (5UI ~ I).

The PCR reaction consists of several amplification cycles that are

described below: 1 stage of 5 minutes at 94 oC, 10 cycles at 94 oC

for 15 seconds, then at 57 degrees for 15 seconds and finally at 72 oC for 30 seconds, then 30 cycles at 94 oC for 15 seconds, then at 52 degrees for 15 seconds and finally at 72 oC for 30 seconds, and an elongation final made at 72 OC for 7

minutes

In order to carry out the selection stage h (in particular h2), of the following eight preferred microsatellite molecular markers (SSRs) and defined by the following SEO ID No. 1 to 16 nucleotide primers, the FAD2, CMAGN73, CUCNITRA markers have been used , T J3, CMBR153 and PALT1:

-

CMAGN73 marker defined by the pair of primers of the following

nucleotide sequences:

5 'ATCCMCTCGACCAAGAAAC 3' (SEO ID No. 1) ie the sense primer and 3 'CAGCTCTACAACMCATCTC 5' (SEO ID No. 2) that is the antisense primer.

-

CMBR153 marker defined by the pair of primers of the following

Nucleotide sequences: 5 'TCAAAGACAAGAAGACCAACCA 3' (SEO ID No. 3) ie the sense primer and 3 'TGTGCTAAGAGAGAGAGAGAAGATIG 5' (SEO ID No. 4) ie the antisense primer.

-

CMBR22 marker defined by the pair of primers of the following

nucleotide sequences: 5 'CCAAMCGACCAAATGTICC 3' (SEO ID No. 5) ie the felt primer

Y

3 'ATACAGACACGCCTICCACC 5' (SEO ID No. 6) that is the antisense primer.

-

CMMP73 marker defined by the pair of primers of the following

nucleotide sequences: 5 'GCACTTIGAGTAAGAAGCAGA 3' (SEO ID N "7) ie the sense primer

Y

3 'GCTGTGAGGTIGACTACGA 5' (SEO ID No. 8) ie the antisense primer.

-

CUCNITRA marker defined by the pair of primers of the following

Nucleotide sequences: 5 'CAAACCATAACTICCAAGG 3' (SEO ID No. 9) ie the sense primer and 3 'GGAGATCGACGAATTIGA 5' (SEO ID No. 10) that is the antisense primer.

-

FAD2 marker defined by the pair of primers of the following

nucleotide sequences:

5 'CACGAGCAGAGAGATAATAAA 3' (SEO ID No. 11) that is the primer

sense

and 3 'CCTGAAAGAGAGGAAGAAGAA 5' (SEO ID No. 12) ie the antisense primer.

-

PATL 1 marker defined by the pair of primers of the following

nucleotide sequences:

5 'TACTCCGCCCTCTCTCTCTC 3' (SEO ID No. 13) ie the sense primer and 3 'CAGGTGCAGAATCTGGTAGT 5' (SEO ID No. 14) ie the antisense primer.

-

Marker T J3 defined by the pair of primers of the following

nucleotide sequences:

5 'TGGGCCTACGCTACAAACTT 3' (SEO ID No. 15) ie the primer

sense

and 3 'AGCAGCACAAAAGCACTTCA 5' (SEO ID No. 16) ie the primer

antisense

These primers have been performed according to the methods known by the

skilled. For given satellite markers, depending on the

size of the fragments amplified by PCR thanks to the specific primers (see tables 3 to 6 that we will see later), the plants H, or DH, or H and DH have been selected. In this way, the markers

Molecules used allow, among other aspects, to determine whether or not a plant is the result of fertilization, whether it is homozygous or not.

If the plant is the result of a fertilization between the male plant material and the female plant material or if it has not undergone reduction

Gamética, is removed after analysis with molecular markers SSR, but if it is a dihaploid plant, it is preserved.

Plants n undergo in vitro colchicine treatment to double their

genome (see stage i) already exposed). Then they are replanted, they

Root and sprout.

Stage i) Genome duplication (colchicine treatment):

5 1-Isolate the main apex of the plant as well as axillary buds if there are (cuttings); 2-Prepare a colchicine solution of 5 per 1000 (0.5 9 colchicine in 100 ml) and sterilize it; 3-Place the cuttings in small sterile Petri dishes (BP 35x10);

10 4-In a microbiological safety cabinet, take a sufficient amount of colchicine to cover the cuttings (6 to 8 ml), then close the plate; 5-Let it soak for 2 hours; 6-Remove the cuttings and rinse them in small boats with sterile water;

15 7-After having dried them slightly on sterile paper, replant all the cuttings in boats in their medium of origin; 8-Replant the developing yolks in the source medium by numbering them from C1 (<<Cl> for colchicine) to CX; 9-When the cuttings have taken root and are therefore ready,

20 cultivate them in vivo in a coconut support with an irrigation of the nutrient solution.

11-Results of fruit set and number in fruits according to genotypes (Table 1) Table 1

Irradiation N "Wave and Time Irradiation Day and Time pollination

1 J-1 16H J 7H 2 J 8H J 11H30 3 J-1 16H J 7H • J 8H J 12H15 5 J 8H J 11H30 Tolal

Number Frulos

VG06

VG06 pollinated Number of frulos 10 9 19 eleven 12 61

general

VG06 term% grip of 10 9 19 eleven 12 61

VG06

100% 100% 100% 100% 100% 100%

VNOO1

Fruits Number

eleven

10 19 fifteen 9 61

general

VN001 pollinated

Term number VN001 at term

eleven 10 16 . B 59

Irradiation N "

one 2 3 • 5 Total

Day and Time irradiation

J-1 16H J aH J-1 16H J aH J aH

Day and Time for Linization

J 7H J 11H30 J 7H J 12H15 J 11H30

% grip of

100%

100%

100%

93% 88% 97%

VNOO1

VG06

VG06 175 Gy JIB 7 7 13 7 7 41

detail

VG06 200 Gy JIB 3 2 • 2 3 .

(Number of fruits)

Controls X X 2 2 2 •

VNOO1 detail

VN001 175 Gy JIB VNOQ1 200 Gy JIB 7 3 7 2 12 • 9 5 6 2 41,.

(Number

Controls

one one X X X 2

of fruits)

Tola l

twenty-one 19 35 30 twenty 125

There is a very good general setting. 127 fertilizations have allowed the development of 125 fruits, that is, 98% success.

5 111-Results obtained based on parental genotypes and the irradiation (ionization) dose performed! Table 2)

Doses 175 Gy and 200 Gy have been tested in the reproductive plant material of the male parent JIB and the results of the gynogenesis with the female plant material (VN001 and VG06) are given below.

Genotype

Gy dose Number of fruits Number of grains Embryos number Size half embryos cm Number of plants in tubes

VG06

175 (H) 200 (0) •• 16 9707 3710 289 30 0.18 0.12 twenty-one

VN001

175 (H) 200 (0) ., 16 2435 722 62 11 0.13 0.1 • OR

Table 2

The table shows that the 200 Gy dose used for irradiation of the

Pollen is too strong to obtain, with genotypes VG06 and VNOO 1,

Embryos that develop. On the contrary, the 175 Gy dose is adapted

better since it allows to produce not only more fruits with the two female genotypes under test (VG06 and VN001), but also more embryos develop (24 for VG06 and 4 for VN001).

IV-Selection with molecular markers

1. Analysis of the plants resulting from VN001

To the Analysis of the plants in order to identify the plants that contain genetic material of the male parent (Table 3):

The VNOOl hybrid plants used as female in the procedure of

Plant production by gynogenesis with irradiation of the male parental reproductive material possess an allele of 171 base pairs

for the FAD2 marker, while JIB plants used as a plant

male have an allele of 169 base pairs for that same marker.

Similarly, the allele of the CMAGN73 marker is 128 base pairs for VN001, while it is 132 for JIB, and the CUCNITRA marker allele is 160 base pairs for VN001, while it is 165 for JIB.

Table 3 shows the molecular analysis of the plants resulting from VN001 according to the method of the present invention and indicates, for each plant under test, the presence or absence of genetic material of the male parent.

Markers

Plant No.

FA02 CMAGN73 CUCNITRA

JIB

169 132 165

VNOO1

171 128 160

Markers

Plant No.

FAD2 CMAGN73 CUCNITRA

1st floor

X X X

2nd floor

X X X

3rd floor

X X

4th floor

X X X X

Table 3

This table 3 shows that the FA02, CMAGN73 and CUCNITRA markers

They allow the identification of plants that have a male genome. After a PCR

5 using the primers (SEO ID N "11 and 12 already defined above) specific to the molecular marker FA02, the analysis of this PCR shows for the plants 1, 3 and 4 the presence of the 169 base pair allele specific to the reproductive material JIB male Plants 1, 3 and 4 therefore have a male genome in their genome.

10 demonstrate therefore that the use of determined molecular marker (s) (here CMAGN73, CUCNITRA and particularly FA02) allows an efficient and safe analysis and selection of the origin of the plants produced.

15 BI Analysis of plants in order to distinguish homozygous plants from heterozygous plants (Table 4):

The VN001 hybrid plants used as female in the process of

plant production by gynogenesis with irradiation of the male parental reproductive material 20 have an allele of 130 base pairs and

an allele of 138 base pairs for the T J3 marker.

Similarly, CMBR153 marker alleles are 166 and 182 pairs

of bases for VN001, and those of PLAT1 are 195 and 204 base pairs.

Table 4 shows the molecular analysis of the plants resulting from VN001 according to the method of the present invention and indicates, for each plant under test, the presence of one or more alleles of each marker.

Markers

Plant No.

TJ3 CMBR153 PALT1

VNOO1

130 138 166 182 195 204

1st floor

X X X X

2nd floor

X X X

3rd floor

X X X X

4th floor

X X X

Table 4

This lip shows that plates 1 and 3 are heterozygous. In fact, thanks to the T J3 marker, the presence of an allele of 130 base pairs 5 and another allele of 138 base pairs is confirmed. With markers T J3, CMBR153 and

PAL T1, plants 2 and 4 are homozygous. This results

demonstrate that the use of determined molecular marker (s) (here T J3, CMBR153, PATL 1) allows an efficient and safe analysis and selection of the homozygous / heterozygous state, and possibly of

10 ploidy, of the plants produced.

2. Analysis of the plants resulting from VG06

To the Analysis of the plants in order to identify the plants that contain genetic material of the male parent (Table 5):

VG06 hybrid plants used as female in the process of plant production by gynogenesis with irradiation of the material

male parental player have an allele of 171 base pairs 20 for the FAD2 marker, while JIB plants used as a plant

male have an allele of 169 base pairs for that same marker.

Similarly, the CUCNITRA marker allele is 160 for VN001, while 165 for JIB, and the CMAGN73 marker allele is 25 128 base pairs for VG06, while it is 132 for JIB.

Table 5 shows the molecular analysis of the plants resulting from VG06 according to the process of the present invention and indicates, for each plant

tested, the presence or absence of genetic material of the

parental male

Marker

Plant No.

FA02 CUCNITRA CMAGN73

JIB

169 165 132

VG06

171 160 128

1st floor

X X X

2nd floor

X

3rd floor

X

4th floor

X X X X X

5th floor

X X X X

6th floor

X X X X X

7th floor

X X X

8th floor

X X X

9th floor

X X X X

10th floor

X X X X X

11th floor

X X X X

12th floor

X X X X X X

13th floor

X X X X

14th floor

X X

15th floor

X X

16th floor

X X

17th floor

X X

18th floor

X X X

19th floor

X X

Table 5

5 In the same way as for analyzes with VN001 (table 3), thanks to the molecular markers FAD2, CUCNITRA, CMAGN73, this table allows

identify plants that contain the male genome: plants 4, 5, 6, 9,

10, 11, 12, 13, 18 and 19. These results therefore demonstrate that the use of molecular marker (s) determined (here) FAD2,

10 CUCNITRA, CMAGN73) allows an efficient and safe analysis and selection of the origin of the plants produced.

BI Analysis of plants in order to distinguish plants homozygous of heterozygous plants (Table 6):

The VG06 hybrid plants used as female in the process of plant production by gynogenesis with irradiation of the male parenlal reproductive material have an allele of 130 base pairs and an allele of 138 base pairs for the T J3 marker.

Similarly, the alleles of the CMBR153 marker are 166 and 182 base pairs 10 for VG06, and those of PLAT1 are 195 and 204 base pairs.

Table 6 shows the molecular analysis of the plants resulting from VG06 according to the procedure of the present invention and indicates, for each plant under test, the presence of one or more alleles of each marker.

Markers

Plant No.

TJ3 CMBR153 PALT1

VG06

130 136 166 162 195 204

1st floor

X X X X X

2nd floor

X X X

3rd floor

X X X X X

4th floor

X X X

5th floor

X X X X X

6th floor

X X X

7th floor

X X X

8th floor

X X X X X

9th floor

X X X X X

10th floor

X X X X X X

11th floor

X X X X

12th floor

X X X X

13th floor

X X X X X X

14th floor

X X X

15th floor

X X X X X

16th floor

X X X X

17th floor

X X X X X

Markers

Plant No.

TJ3 CMBR153 PAlT1

VG06

130 13B 166 1B2 195 204

18th floor

X X X X

19th floor

X X X X X

Table 6

This table 6 shows that with markers T J3, CMBR153 and PAL T1, only plants 4 and 6 are homozygous, the other plants are shown

5 heterozygotes with these molecular markers. These results demonstrate that the use of a determined molecular marker (s) (here T J3, CMBR153, PAL T1) allows an efficient and safe analysis and selection of the homozygous / heterozygous state, and possibly ploidia, of the plants produced.

IV-Summary table of the effect of irradiation doses on

Obtaining heterocyclotic plants and haploid plants (Table 7)

Irradiation dose

Year Number of plants tested by flow cytometry and SSR Number of heterozygous plants (2 ") Number of haploid plants (n)

25Gy / 150Gy

2003 62B 62B OR

125 Gy f 150 Gy

2004 1734 1734 OR

125Gy

2005 2931 2931 OR

125 Gy 1150 Gy

2006 461 460 OR

175 Gy {200 Gy

2007 twenty 10 10

Table 7

15 Doses of 25 to 50 Gy involve the production of numerous plants, all identified as heterozygous. The dose of 175 Gy allows not only to reduce the number of plants produced to be analyzed, but also to considerably increase the number of haploid plants among the plants

20 produced. As previously explained, these haploid plants are of special interest. Consequently, this table demonstrates that the irradiation dose influences the success of the method applied in the present invention and that this dose can be optimized for increased production of haploid plants. Thus, with the dose of 175 Gy it is possible to improve the production of haploid H and double plants

5 HO haploids, by ginogenesis induced by an irradiated male gametophyte and consequently obtain a genome stabilization of the plants produced with a reduced number of generations.

VI-Culture media

Composition

S1C 53. 52 54

TZ2

X % X

TZ4

X % X

Saccharose

30 911 59 " 59 "

Plan! Preservation Mixture (Kalys®)

1 mili 1 mili 1 mili

Vitro Agar

89 " 89 " 89 " 89 "

pH

5.9 5.9 5.9 5.9

Autoclave 120 OCf20 min

Quinetina

0.05 mgll 0.05 mgll

Acetic Naphthalene Acid

0.05 mgll 0.05 mgll

Cefotaxime

300 mgll

Table 8

Middle combosition

TZ2 TZ4

Macro Murashige the Skoog

X 5

CP macro

X

Micro Murashige the Skoog

X

Micro CP

X

MOREL Vitamins

X

Vitamins P

X

Saccharose

3% 10

Table 9

15 Details of the compositions of table 9: -TZ4 medium without sucrose or agar:

CP element macro: in mg. L "KN0 3 2150 NH4N0 3 1238 Ca (N0 3) 2, 4 H20 50 CaCI2,2H20 313 MgS04,7H20 412 KCI 7 Na H2P0 4, H20 38 KH2P0 4 142 (N H4), S04 34

Fer EDTA: in mg. L "FeS04.7H20 27.8 Na2EDTA 37.8

CP element macro: in mg. L "MnS04, H20 22.13 H3B0 3 3.15 ZnS04.7H2O 3.62 Na2Mo04 0.188 CuS04.5H2O 0.016 CoCI2 0.016 KI 0.695

Vitamins P: in f..lQ L-1

Meso Inositol 50.35 mg. L- 'Thiamine 600 ug. L - 'Nicotic acid 700 Pyridoxine 5500 Calcium pantothenate 500 Biotin 5

-

TZ2 medium with sucrose:

Micro element MS: in rng. L "1

KN0 3 1900

NH4N0 3 1650 CaC !, 332.02 Mg SO "180.54 KH, P0 4 170 FeNa EDTA: 36.70

Macro element MS: in mg, L "MnSO" H, O 16.90 H3B0 3 6.20 ZnSO ,, 7H, 0 8.60 Na, MoO "2H, 0 0.25 CuSO ,, 5H, 0 0.025 CoCI, , 6H, 0 0.025 KI 0.83

MOREL Vitamins: in mg, L- 'Meso Inositol 100 mg. L "Thiamine 1

Nicotinic acid 1 Pyridoxine 1

Calcium pantothenate 1 Biotin 0.01 Sucrose: 30000.00 rngll

Claims (17)

1. Production procedure of haploid H, double haploid HO and / or dihaploid OH plants, the HO and OH being homozygous or essentially homozygous, this being a type of procedure that corresponds to the technique of gynogenesis induced by irradiated pollen, characterized in that it comprises:

-

an irradiation stage of the male parental reproductive material with a dose between 160 and 190 gamma rays, preferably between approximately 165 and 185 gamma rays (Gy), or a selection stage of haploid plants H, or OH, or HY OH through the use of molecular marker (s), - or a stage of irradiation of the male parental reproductive plant material with a dose between 165 and 185 gamma rays (Gy) and

a stage of selection of haploid plants H, or DH, or H and OH by the use of molecular marker (s). 2. Procedure according to claim 1, characterized in that it comprises the following successive stages: a) Apply male parental reproductive material; b) Irradiate said male parental reproductive material with a dose between 160 and 190 gamma rays (Gy), preferably between 165 and 185 gamma rays; e) Pollinating the hybrid female parental reproductive material with said reproductive material of the irradiated male parent; d) Collect fruits whose grains contain embryos; e) Extract the grains of said fruits; f) Remove the embryos from said grains; g) Put the embryos in culture in a suitable medium until obtaining of a plant. 3. Method according to claim 2, characterized in that it also comprises, after step g), a step h) which consists of selecting the haploid plants H, or OH, or HY OH by flow cytometry, or by the use of molecular marker (s), or by cytometry of flow and use of molecular marker (s). 4. Method according to claim 3, characterized in that it comprises in step h of selection of haploid plants H, or OH, or H and OH: -1-the use of specific molecular marker (s) of given allele (s) contained in the genetic material of the irradiated male parent to select from among the plants obtained after stage g ) plants that do not contain irradiated male parental genetic material or that they have an insufficient quantity to cause the offspring of these plants a disjunction of a phenotypic and / or genotypic character; -11-the use of specific molecular marker (s) of given allele (s) contained in the genetic material of the hybrid female parent, allowing this (these) marker (s) to determine the homozygous / heterozygous state of the plants to be selected, that is, the plants obtained after step g), using this (these) marker (s) to select homozygous or essentially homozygous plants, in particular OH plants. 5. Method according to any of the preceding claims, characterized in that it also comprises a stage i) of duplication of the genome of haploid H plants. 6. Method according to claim 5, characterized in that the step i) Genome duplication is performed using colchicine. 7. Method, in particular according to any one of the preceding claims, of production of haploid H, double haploid HO and / or dihaploid OH plants, the HO and OH being homozygous or essentially homozygous, by irradiation-induced gynogenesis of the male parental reproductive material, characterized in that it comprises a previous stage of determining the (or the) adequate irradiation dose (s) to increase the yields of said plants based on multiple factors given and preferably selected from among the plant species, the male parental genotypes and of the female parent, the climatic and physiological conditions, the time of harvest of the fruits, the level of growth of the embryos collected with a view to their cultivation, the level of development of embryos placed in culture; consisting of this previous stage: i) in testing, for a given factor, different doses of irradiation on the reproductive plant malerial of the male parent, ii) in applying, for each irradiation dose tested, a step h ') of selection of the plants H, or OH, or HY OH, including said step h': -1-the use of molecular marker (s) specific (s) of allele (s) of the irradiated male parental genetic material to select from plants obtained after irradiated pollen-induced gynogenesis plants that do not contain irradiated male parental plant material or that possess an insufficient amount to cause, in the offspring of said plants, a disjunction of a phenotypic and / or genotypic character; -11-the use of specific molecular marker (s) of given specific allele (s) of the genetic material of the hybrid female parental applied in gynogenesis, allowing this (these) marker (s) to determine the homozygous / heterozygous state of the plants to be selected, that is, of the plants obtained after gynogenesis, using this (these) marker (s) to select homozygous plants or essentially homozygous, in particular OH plants. iii) in counting, for each dose of irradiation tested, haploid H plants,

or OH, or H AND OH obtained; iv) to calculate, for each irradiation dose tested and from the counts resulting from (iii), the yield in haploid plants H, or OH,

or H and OH obtained;

v) in comparing the counts resulting from (jii) and / or yields resulting from (iv) to deduce the appropriate irradiation dose (s). 8. Method according to any of the preceding claims, characterized in that the parental plants are selected from the genus Cucurbita Method according to any of the preceding claims, characterized in that the molecular marker (s) used is (are) a microsatellite marker (s) (SSR). 5. 10. Method according to any of the preceding claims, characterized in that the molecular marker (s) used are select (n) from the markers defined by one or some pairs of SEO ID nucleotide sequence primers No. 1 to 16, preferably SEO ID No. 3 to 14. eleven . Use of a molecular marker characterized in that it is a microsatellite marker (S SR) selected from the markers defined by one or some pairs of nuc1eotidic sequence primers attached SEO ID No. 1 to 16, preferably SEO ID No. 3 to 14, in the method according to any one of claims 1 to 10. 12. Haploid H embryos of plants such as those obtained by the procedure as defined in any of claims 1 to 10. 13. OH dihaploid embryos of plants such as those obtained by the procedure as defined in any of the claims 1 to 10 14. Offspring of the plants obtained by the procedure as is defined in any one of claims 1 to 10. 15. Offspring of the wafer plants by the procedure as is defined in any of claims 1 to 10, obtaining said offspring by crossing between two plants (HD or OH) resulting from the procedure and as defined in any of the claims 1 to 10, or by crossing between a HO or OH plant resulting from the procedure and as defined in any of claims 1 to 10 and a plant resulting from a stable line or resulting from repeated self-fertilization. 16. Plant seeds obtained by the process as defined in any one of claims 1 to 10 or from the embryos as defined in any of the claims 12 or 13, as well as from plans result from the offspring as defined in any of the re vindications 14 015.